Nicotinic receptors regulate the survival of newborn neurons in the adult olfactory bulb

Abstract

Cholinergic axons and nicotinic receptors are abundant in all layers of the olfactory bulb (OB), the main region of newborn neuron integration in the adult brain. Here, we report that the OB granule cell layer in mice lacking the predominant form of brain high-affinity nicotinic acetylcholine receptors (beta(2)(-/-) mice) displayed nearly 50% more newborn neurons and significantly fewer apoptotic cells than did beta(2)(+/+) mice. Conversely, in vivo chronic nicotine exposure significantly decreased the number of newborn granule cells in beta(2)(+/+) but not beta(2)(-/-) adult mice, confirming that the survival of newborn neurons can be controlled by the activation of beta(2)-containing nicotinic acetylcholine receptors. Unexpectedly, investigating the behavioral consequence of an increased number of granule cells in beta(2)(-/-) mice revealed that these animals have a less robust short-term olfactory memory than their wild-type counterparts. Taken together, these results provide evidence that high-affinity nicotinic receptors are involved in the maturation of adult OB local circuits. They also indicate that an increase in the number of granule cells does not necessarily correlate with better olfactory performance and further highlight the importance of cholinergic afferents for olfactory processing.

Fig. 1.

Increased survival of newborn neurons in olfactory bulb of adult β₂^-/- mice. (a) ¹²⁵I-EB binding to high-affinity nAChRs in the OB of adult β₂^+/+ (Left) and β₂^-/- mice (Right). Note the high levels of binding in the granule cell layer (gcl) and glomerular layer (gl) of WT mice. Such binding could not be detected (nd) in the gcl and was reduced by 88% in the gl of β₂^-/- mice (b). (c, e, and g) Representative micrographs illustrating the layers in which densities of BrdUrd+ cells were quantified 21 days after repeated BrdUrd injections, i.e., the granule cell layer and glomerular layer of the OB and the granular layer of the dentate gyrus (dg), in both β₂^+/+ and β₂^-/- mice (Left and Right, respectively). (d, f, and h) Densities of BrdUrd+ cells determined from the gcl, gl, and dg, respectively. The only statistically significant difference between genotypes was found for the gcl, in which β₂^-/- mice had on average 46% more granule cells than their WT counterparts. ^**, P < 0.01, by Student's t test; n = 6 per genotype, from two independent experiments. (Scale bars: a, 500 μm; c and g, 100 μm; e, 25 μm.)

Fig. 2.

Proliferation is not affected in β₂^-/- mice. Representative micrographs of BrdUrd-immunostained sections of SVZ (a) and RMS (c) in β₂^+/+ and β₂^-/- mice (Left and Right, respectively), and for which densities of BrdUrd+ profiles were determined 2 h after a single injection of BrdUrd (b and d). lv, lateral ventricle; cc, corpus callosum; str, striatum. (Scale bars: 100 μm.)

Fig. 3.

The fate of newborn cells is not affected in β₂^-/- mice. (a and b) Confocal microscopy images of BrdUrd (red) and NeuN (green) double-immunofluorescent labeling in the OB granule cell layer, 21 days after BrdUrd injections (n = 3 per genotype). Reconstructed orthogonal projections are presented as viewed in the x-z (Upper) and y-z (Right) planes. The proportion of double-labeled (yellow) cells did not differ between β₂^+/+ and β₂^-/- mice (b), as determined from the analysis of almost 1,000 cells per group. (c and d) Decreased apoptosis in the granule cell layer of β₂^-/- mice. (c) TUNEL staining in granule cell layer sections from adult β₂^+/+ (Left) and β₂^-/- mice (Right). Arrows point to apoptotic cells. (d) Significantly fewer apoptotic profiles were found in β₂^-/- mice. β₂^+/+, n = 3, and β₂^-/-, n = 4; ^*, P < 0.05 by Student's t test. (Scale bars: a, 10 μm; c, 25 μm.)

Fig. 4.

Chronic nicotine exposure decreases survival of newborn OB neurons via ^*β₂-nAChR activation. (a and c) Representative micrographs of BrdUrd+ cells in the granule cell layer of chronic saline- and nicotine-exposed β₂^+/+ and β₂^-/- mice, and corresponding densities of BrdUrd+ cells (b and d). β₂^+/+, n = 6 per group; β₂^-/-, n = 3 per group. ^*, P < 0.05 by Student's t test. (Scale bars: 100 μm.)

Fig. 5.

Same detection threshold but decreased olfactory memory in β₂^-/- mice. (a) Time spent investigating (sniffing or rearing) 10^-3 M dilutions of 10 different simple odors. Data represent the ratio of time spent investigating odors during second compared with first presentation. Animals, odors, and time intervals were randomly tested over the 4-week period of experimentation. Whereas β₂^+/+ mice spent significantly less time investigating odors after up to 240 min (Left), β₂^-/- mice did not (Right). Animals were retested for this time interval (white columns), and the difference between groups was confirmed. (b) Time spent by mice investigating a simple odor of increasing concentration. Mineral oil (control) or diluted solutions of heptanol were presented to mice on consecutive 5-min periods. A single heptanol concentration was used each day, from the least to the most concentrated. Both genotypes detected heptanol at 10^-4 M. β₂^+/+, n = 15, and β₂^-/-, n = 16; ^**, P < 0.01 and ^*, P < 0.05, with Wilcoxon's test.